Since high-density lipoprotein (HDL) receives cholesterol from various tissues including walls of blood vessels with arteriosclerosis, it is involved in the action of efflux of cholesterol accumulated in cells. Therefore, HDL cholesterol is also called the reverse cholesterol transport system. High-density lipoprotein is known to have negative correlation with arteriosclerotic diseases such as coronary arteriosclerosis. Accordingly, an HDL value lower than a predetermined lower limit is regarded as an indication of hyperlipidemia, and the value is known to be useful as an index of arteriosclerosis.
HDL is constituted by apoprotein, phospholipid, cholesterol and neutral fat. HDL has a density of d=1.063 to 1.210 g/mL, and can be divided into two fractions, that is, HDL2, wherein d=1.063 to 1.125 g/mL, and HDL3, wherein d=1.125 to 1.210 g/mL. A notch is found at the portion of d=1.125 g/mL in the distribution curve of lipoprotein, and the part having higher densities in the curve corresponds to HDL3. Alternatively, HDL can be divided into subfractions based on the content of apolipoprotein E among the apoproteins in HDL, and HDLs having higher contents of apoE are defined as apoE-rich HDLs.
Conventionally, HDL is known to function not only as a whole but also as the individual HDL2 and HDL3 subfractions having different functions. HDL2 is known to have an antiatherogenic action. It is clinically known that CETP deficiency prevents metabolism of HDL to LDL or IDL, leading to an increase in the HDL cholesterol level. It is also said that the HDL increased by CETP deficiency is HDL2. Further, it is also said that CETP deficiency causes an increase in apoE-rich HDL, and that, since Apo-E-rich HDL has a strong cholesterol-efflux ability and antiplatelet action, it is a better HDL among HDLs. Further, a decrease in the hepatic lipase activity prevents conversion of HDL3 to HDL2, resulting in an increase in HDL3. That is, the ratios of subfractions of HDL may vary depending on the clinical condition, and measurement of HDL alone hardly allows detection of such changes. In view of such tendencies, it is expected that measurement of each of the HDL subfractions may contribute to judgment of whether or not a patient is suffering from a disease such as arteriosclerosis or hyperlipidemia, and of the cause of the disease. Further, at present, in view of these functions of HDL subfractions, manufacturers are developing therapeutic agents that inhibit the function of CETP, decrease the LDL cholesterol level, and increase the HDL cholesterol level.
Establishment of a simple method for measuring the HDL subfractions may lead to detailed elucidation of their functions, and to their therapeutic effects in the future.
Examples of the methods for measuring HDL subfractions that are known at present include ultracentrifugation, high-performance liquid chromatography (HPLC), HDL3 precipitation (Patent Document 1) and NMR.
In ultracentrifugation, fractionation is carried out utilizing the difference in the density of lipoprotein. This method has drawbacks in that the operation requires a skill; the method takes many days; and the cost is high. In the method by Okazaki et al. wherein HPLC is used for separating HDL2 and HDL3, the operation takes a long time, and special equipment is required. HDL3 precipitation is a method wherein a reagent containing a divalent metal ion and dextran sulfate is used to aggregate lipoproteins other than HDL3, and HDL3 in the supernatant portion is recovered by centrifugation and measured using an automatic analyzer. This method is not widely used since the method has drawbacks in that the operation of this method also requires a skill; the method is a manual method; the method requires an operation of sample pretreatment; and a certain length of time is required before measurement. Further, NMR, which is a method wherein the number of particles of lipoprotein is measured by nuclear magnetic resonance, is not commonly employed since the method requires special equipment.
There is a method for analyzing HDL subfractions (Patent Document 2). Although this method enables measurement with a general purpose automatic apparatus, the method employs a method wherein a surfactant is used to prevent an enzyme from acting on lipoproteins other than HDL3. By measuring HDL3 and subtracting the value of HDL3 from total HDL, HDL2 can be measured, but the method does not directly measure HDL2.
Thus, as an alternative to the above methods, a reagent that enables simple and more selective quantification of cholesterol in HDL2 needs to be invented.